Alterations in the long QT syndrome genes KVLQT1 and SCN5A and methods for detecting same

ABSTRACT

Long QT Syndrome (LQTS) is a cardiovascular disorder characterized by prolongation of the QT interval on electrocardiogram and presence of syncope, seizures and sudden death. Five genes have been implicated in Romano-Ward syndrome, the autosomal dominant form of LQTS. These genes are KVLQT1, HERG, SCN5A, KCNE1 and KCNE2. Mutations in KVLQT1 and KCNE1 also cause the Jervell and Lange-Nielsen syndrome, a form of LQTS associated with deafness, a phenotypic abnormality inherited in an autosomal recessive fashion. Mutational analyses were used to screen 262 unrelated individuals with LQTS for mutations in the five defined genes. A total of 134 mutations were observed of which eighty were novel.

CROSS REFERENCE TO RELATED APPLICATIONS

The present invention is a division of U.S. patent application Ser. No.09/840,125 filed 24 Apr. 2001 which in turn is a division of U.S. patentapplication Ser. No. 09/634,920 filed 9 Aug. 2000. Ser. No. 09/634,920is related and claims priority under 35 U.S.C. § 119(e) to provisionalpatent application Ser. No. 60/190,057 filed 17 Mar. 2000 and toprovisional patent application Ser. No. 60/147,488 filed 9 Aug. 1999.Each application is incorporated herein by reference.

This application was made with Government support from NHLBI under GrantNos. RO1-HL46401, RO1-HL33843, RO1-HL51618, P50-HL52338 andMO1-RR000064. The federal government may have certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Long QT Syndrome (LQTS) is a cardiovascular disorder characterized byprolongation of the QT interval on electrocardiogram and presence ofsyncope, seizures and sudden death, usually in young, otherwise healthyindividuals (Jervell and Lange-Nielsen, 1957; Romano et al., 1963; Ward,1964). The clinical features of LQTS result from episodic ventriculartachyarrhythmias, such as torsade de pointes and ventricularfibrillation (Schwartz et al., 1975; Moss et al., 1991). Two inheritedforms of LQTS exist. The more common form, Romano-Ward syndrome (RW), isnot associated with other phenotypic abnormalities and is inherited asan autosomal dominant trait with variable penetrance (Roman et al.,1963; Ward, 1964). Jervell and Lange-Nielsen syndrome (JLN) ischaracterized by the presence of deafness, a phenotypic abnormalityinherited as an autosomal recessive trait (Jervell and Lange-Nielsen,1957). LQTS can also be acquired, usually as a result of pharmacologictherapy.

In previous studies, we mapped LQTS loci to chromosomes 11p15.5 (LQT1)(Keating et al., 1991), 7 q35-36 (LQT2) (Jiang et al., 1994) and LQT3 to3p21-24 (Jiang et al., 1994). A fourth locus (LQT4) was mapped to4q25-27 (Schott et al., 1995). Five genes have been implicated inRomano-Ward syndrome, the autosomal dominant form of LQTS. These genesare KVLQT1 (LQT1) (Wang Q. et al., 1996a), HERG (LQT2) (Curran et al.,1995), SCN5A (LQT3) (Wang et al., 1995a), and two genes located at21q22-KCNE1 (LQT5) (Splawski et al., 1997a) and KCNE2 (LQT6) (Abbott etal., 1999). Mutations in KVLQT1 and KCNE1 also cause the Jervell andLange-Nielsen syndrome, a form of LQTS associated with deafness, aphenotypic abnormality inherited in an autosomal recessive fashion.

KVLQT1, HERG, KCNE1 and KCNE2 encode potassium channel subunits. FourKVLQT1 α-subunits assemble with minK (β-subunits encoded by KCNE1,stoichiometry is unknown) to form I_(K) channels underlying the slowlyactivating delayed rectifier potassium current in the heart (Sanguinettiet al., 1996a; Barhanin et al., 1996). Four HERG α-subunits assemblewith MiRP1 (encoded byKCNE2, stoichiometry unknown) to form I_(Kr)channels, which underlie the rapidly activating, delayed rectifierpotassium current (Abbott et al., 1999). Mutant subunits lead toreduction of I_(Ks) or I_(Kr) by a loss-of-function mechanism, oftenwith a dominant-negative effect (Chouabe et al., 1997; Shalaby et al.,1997; Wollnik et al., 1997; Sanguinetti et al., 1996b). SCN5A encodesthe cardiac sodium channel that is responsible for I_(Na), the sodiumcurrent in the heart (Gellens et al., 1992). LQTS-associated mutationsin SCN5A cause a gain-of-function (Bennett et al., 1995; Dumaine et al.,1996). In the heart, reduced I_(Ks) or I_(Kr) or increased I_(Na) leadsto prolongation of the cardiac action potential, lengthening of the QTinterval and increased risk of arrhythmia. KVLQT1 and KCNE1 are alsoexpressed in the inner ear (Neyroud et al., 1997; Vetter et al., 1996).Others and we demonstrated that complete loss of I_(Ks) causes thesevere cardiac phenotype and deafness in JLN (Neyroud et al., 1997;Splawski et al., 1997b; Tyson et al., 1997; Schulze-Bahr et al., 1997).

Presymptomatic diagnosis of LQTS is currently based on prolongation ofthe QT interval on electrocardiogram. Genetic studies, however, haveshown that diagnosis based solely on electrocardiogram is neithersensitive nor specific (Vincent et al., 1992; Priori et al., 1999).Genetic screening using mutational analysis can improve presymptomaticdiagnosis. However, a comprehensive study identifying and cataloging allLQTS-associated mutations in all five genes has not been achieved. Todetermine the relative frequency of mutations in each gene, facilitatepresymptomatic diagnosis and enable genotype-phenotype studies, wescreened a pool of 262 unrelated individuals with LQTS for mutations inthe five defined genes. The results of these studies are presented inthe Examples below.

The present invention relates to alterations in the KVLQT1, HERG, SCN5A,KCNE1 and KCNE2 genes and methods for detecting such alterations.

The publications and other materials used herein to illuminate thebackground of the invention or provide additional details respecting thepractice, are incorporated by reference, and for convenience arerespectively grouped in the appended List of References.

The present invention is directed to alterations in genes and geneproducts associated with long QT syndrome and to a process for thediagnosis and prevention of LQTS. LQTS is diagnosed in accordance withthe present invention by analyzing the DNA sequence of the KVLQT1, HERG,SCN5A, KCNE1 or KCNE2 gene of an individual to be tested and comparingthe respective DNA sequence to the known DNA sequence of the normalgene. Alternatively, these genes of an individual to be tested can bescreened for mutations which cause LQTS. Prediction of LQTS will enablepractitioners to prevent this disorder using existing medical therapy.

SUMMARY OF THE INVENTION

The present invention relates to alterations in the KVLQT1, HERG, SCN5A,KCNE1 and KCNE2 genes and methods for detecting such alterations. Thealterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes includemutations and polymorphisms. Included among the mutations areframeshift, nonsense, splice, regulatory and missense mutations. Anymethod which is capable of detecting the alterations described hereincan be used. Such methods include, but are not limited to, DNAsequencing, allele-specific probing, mismatch detection, single strandedconformation polymorphism detection and allele-specific PCRamplification.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the predicted topology of KVLQT1and the locations of LQTS-associated mutations. KVLQT1 consists of sixputative transmembrane segments (S1 to S6) and a pore (Pore) region.Each circle represents an amino acid. The approximate location ofLQTS-associated mutations identified in our laboratory are shown withfilled circles.

FIG. 2 is a schematic representation of HERG mutations. HERG consists ofsix putative transmembrane segments (S1 to S6) and a pore (Pore) region.Location of LQTS-associated mutations are shown with filled circles.

FIG. 3 is a schematic representation of SCN5A and locations ofLQTS-associated mutations. SCN5A consists of four domain (DI to DIV),each of which has six putative transmembrane segments (S1 to S6) and apore (Pore) region. Location of LQTS-associated mutations identified inour laboratory are shown with filled circles.

FIG. 4 is a schematic representation of minK and locations ofLQT-associated mutations. MinK consists of one putative transmembranedomain (S1). The approximate location of LQTS-associated mutationsidentified in our laboratory are shown with filled circles.

FIG. 5 is a schematic representation of the predicted topology of MiRP1and locations of arrhythmia-associated mutations. MiRP1 consists of oneputative transmembrane domain (S1). The approximate location ofarrhythmia-associated mutations identified in our laboratory are shownwith filled circles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to alterations in the KVLQT1, HERG, SCN5A,KCNE1 and KCNE2 genes and methods for detecting such alterations. Thealterations in the KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 genes includemutations and polymorphisms. Included among the mutations areframeshift, nonsense, splice, regulatory and missense mutations. Anymethod which is capable of detecting the mutations and polymorphismsdescribed herein can be used. Such methods include, but are not limitedto, DNA sequencing, allele-specific probing, mismatch detection, singlestranded conformation polymorphism detection and allele-specific PCRamplification.

KVLQT1, HERG, SCN5A, KCNE1 and KCNE2 mutations cause increased risk forLQTS. Many different mutations occur in KVLQT1, HERG, SCN5A, KCNE1 andKCNE2. In order to detect the presence of alterations in the KVLQT1,HERG, SCN5A, KCNE1 and KCNE2 genes, a biological sample such as blood isprepared and analyzed for the presence or absence of a given alterationof KVLQT1, HERG, SCN5A, KCNE1 or KCNE2. In order to detect the increasedrisk for LQTS or for the lack of such increased risk, a biologicalsample is prepared and analyzed for the presence or absence of a mutantallele of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2. Results of these testsand interpretive information are returned to the health care providerfor communication to the tested individual. Such diagnoses may beperformed by diagnostic laboratories or, alternatively, diagnostic kitsare manufactured and sold to health care providers or to privateindividuals for self-diagnosis.

The presence of hereditary LQTS may be ascertained by testing any tissueof a human for mutations of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2gene. For example, a person who has inherited a germline HERG mutationwould be prone to develop LQTS. This can be determined by testing DNAfrom any tissue of the person's body. Most simply, blood can be drawnand DNA extracted from the cells of the blood. In addition, prenataldiagnosis can be accomplished by testing fetal cells, placental cells oramniotic cells for mutations of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2gene. Alteration of a wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2allele, whether, for example, by point mutation or deletion, can bedetected by any of the means discussed herein.

There are several methods that can be used to detect DNA sequencevariation. Direct DNA sequencing, either manual sequencing or automatedfluorescent sequencing can detect sequence variation. Another approachis the single-stranded conformation polymorphism assay (SSCP) (Orita etal., 1989). This method does not detect all sequence changes, especiallyif the DNA fragment size is greater than 200 bp, but can be optimized todetect most DNA sequence variation. The reduced detection sensitivity isa disadvantage, but the increased throughput possible with SSCP makes itan attractive, viable alternative to direct sequencing for mutationdetection on a research basis. The fragments which have shifted mobilityon SSCP gels are then sequenced to determine the exact nature of the DNAsequence variation. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE) (Sheffield et al., 1991),heteroduplex analysis (HA) (White et al., 1992) and chemical mismatchcleavage (CMC) (Grompe et al., 1989). None of the methods describedabove will detect large deletions, duplications or insertions, nor willthey detect a regulatory mutation which affects transcription ortranslation of the protein. Other methods which might detect theseclasses of mutations such as a protein truncation assay or theasymmetric assay, detect only specific types of mutations and would notdetect missense mutations. A review of currently available methods ofdetecting DNA sequence variation can be found in a recent review byGrompe (1993). Once a mutation is known, an allele specific detectionapproach such as allele specific oligonucleotide (ASO) hybridization canbe utilized to rapidly screen large numbers of other samples for thatsame mutation. Such a technique can utilize probes which are labeledwith gold nanoparticles to yield a visual color result (Elghanian etal., 1997).

A rapid preliminary analysis to detect polymorphisms in DNA sequencescan be performed by looking at a series of Southern blots of DNA cutwith one or more restriction enzymes, preferably with a large number ofrestriction enzymes. Each blot contains a series of normal individualsand a series of LQTS cases. Southern blots displaying hybridizingfragments (differing in length from control DNA when probed withsequences near or including the HERG locus) indicate a possiblemutation. If restriction enzymes which produce very large restrictionfragments are used, then pulsed field gel electrophoresis (PFGE) isemployed.

Detection of point mutations may be accomplished by molecular cloning ofthe KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 alleles and sequencing thealleles using techniques well known in the art. Also, the gene orportions of the gene may be amplified, e.g., by PCR or otheramplification technique, and the amplified gene or amplified portions ofthe gene may be sequenced.

There are six well known methods for a more complete, yet stillindirect, test for confirming the presence of a susceptibilityallele: 1) single stranded conformation analysis (SSCP) (Orita et al.,1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell etal., 1990; Sheffield et al., 1989); 3) RNase protection assays(Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specificoligonucleotides (ASOs) (Conner et al., 1983); 5) the use of proteinswhich recognize nucleotide mismatches, such as the E. coli mutS protein(Modrich, 1991); and 6) allele-specific PCR (Ruano and Kidd, 1989). Forallele-specific PCR, primers are used which hybridize at their 3′ endsto a particular KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 mutation. If theparticular mutation is not present, an amplification product is notobserved. Amplification Refractory Mutation System (ARMS) can also beused, as disclosed in European Patent Application Publication No.0332435 and in Newton et al., 1989. Insertions and deletions of genescan also be detected by cloning, sequencing and amplification. Inaddition, restriction fragment length polymorphism (RFLP) probes for thegene or surrounding marker genes can be used to score alteration of anallele or an insertion in a polymorphic fragment. Such a method isparticularly useful for screening relatives of an affected individualfor the presence of the mutation found in that individual. Othertechniques for detecting insertions and deletions as known in the artcan be used.

In the first three methods (SSCP, DGGE and RNase protection assay), anew electrophoretic band appears. SSCP detects a band which migratesdifferentially because the sequence change causes a difference insingle-strand, intramolecular base pairing. RNase protection involvescleavage of the mutant polynucleotide into two or more smallerfragments. DGGE detects differences in migration rates of mutantsequences compared to wild-type sequences, using a denaturing gradientgel. In an allele-specific oligonucleotide assay, an oligonucleotide isdesigned which detects a specific sequence, and the assay is performedby detecting the presence or absence of a hybridization signal. In themutS assay, the protein binds only to sequences that contain anucleotide mismatch in a heteroduplex between mutant and wild-typesequences.

Mismatches, according to the present invention, are hybridized nucleicacid duplexes in which the two strands are not 100% complementary. Lackof total homology may be due to deletions, insertions, inversions orsubstitutions. Mismatch detection can be used to detect point mutationsin the gene or in its mRNA product. While these techniques are lesssensitive than sequencing, they are simpler to perform on a large numberof samples. An example of a mismatch cleavage technique is the RNaseprotection method. In the practice of the present invention, the methodinvolves the use of a labeled riboprobe which is complementary to thehuman wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene codingsequence. The riboprobe and either mRNA or DNA isolated from the personare annealed (hybridized) together and subsequently digested with theenzyme RNase A which is able to detect some mismatches in a duplex RNAstructure. If a mismatch is detected by RNase A, it cleaves at the siteof the mismatch. Thus, when the annealed RNA preparation is separated onan electrophoretic gel matrix, if a mismatch has been detected andcleaved by RNase A, an RNA product will be seen which is smaller thanthe full length duplex RNA for the riboprobe and the mRNA or DNA. Theriboprobe need not be the full length of the mRNA or gene but can be asegment of either. If the riboprobe comprises only a segment of the mRNAor gene, it will be desirable to use a number of these probes to screenthe whole mRNA sequence for mismatches.

In similar fashion, DNA probes can be used to detect mismatches, throughenzymatic or chemical cleavage. See, e.g., Cotton et al., 1988; Shenk etal., 1975; Novack et al., 1986. Alternatively, mismatches can bedetected by shifts in the electrophoretic mobility of mismatchedduplexes relative to matched duplexes. See, e.g., Cariello, 1988. Witheither riboprobes or DNA probes, the cellular mRNA or DNA which mightcontain a mutation can be amplified using PCR (see below) beforehybridization. Changes in DNA of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2gene can also be detected using Southern hybridization, especially ifthe changes are gross rearrangements, such as deletions and insertions.

DNA sequences of the KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene which havebeen amplified by use of PCR may also be screened using allele-specificprobes. These probes are nucleic acid oligomers, each of which containsa region of the gene sequence harboring a known mutation. For example,one oligomer may be about 30 nucleotides in length, corresponding to aportion of the gene sequence. By use of a battery of suchallele-specific probes, PCR amplification products can be screened toidentify the presence of a previously identified mutation in the gene.Hybridization of allele-specific probes with amplified KVLQT1, HERG,SCN5A, KCNE1 or KCNE2 sequences can be performed, for example, on anylon filter. Hybridization to a particular probe under high stringencyhybridization conditions indicates the presence of the same mutation inthe tissue as in the allele-specific probe.

The newly developed technique of nucleic acid analysis via microchiptechnology is also applicable to the present invention. In thistechnique, literally thousands of distinct oligonucleotide probes arebuilt up in an array on a silicon chip. Nucleic acid to be analyzed isfluorescently labeled and hybridized to the probes on the chip. It isalso possible to study nucleic acid-protein interactions using thesenucleic acid microchips. Using this technique one can determine thepresence of mutations or even sequence the nucleic acid being analyzedor one can measure expression levels of a gene of interest. The methodis one of parallel processing of many, even thousands, of probes at onceand can tremendously increase the rate of analysis. Several papers havebeen published which use this technique. Some of these are Hacia et al.,1996; Shoemaker et al., 1996; Chee et al., 1996; Lockhart et al., 1996;DeRisi et al., 1996; Lipshutz et al., 1995. This method has already beenused to screen people for mutations in the breast cancer gene BRCA1(Hacia et al., 1996). This new technology has been reviewed in a newsarticle in Chemical and Engineering News (Borman, 1996) and been thesubject of an editorial (Editorial, Nature Genetics, 1996). Also seeFodor (1997).

The most definitive test for mutations in a candidate locus is todirectly compare genomic KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 sequencesfrom patients with those from a control population. Alternatively, onecould sequence messenger RNA after amplification, e.g., by PCR, therebyeliminating the necessity of determining the exon structure of thecandidate gene.

Mutations from patients falling outside the coding region of KVLQT1,HERG, SCN5A, KCNE1 or KCNE2 can be detected by examining the non-codingregions, such as introns and regulatory sequences near or within thegenes. An early indication that mutations in non-coding regions areimportant may come from Northern blot experiments that reveal messengerRNA molecules of abnormal size or abundance in patients as compared tocontrol individuals.

Alteration of KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 mRNA expression can bedetected by any techniques known in the art. These include Northern blotanalysis, PCR amplification and RNase protection. Diminished mRNAexpression indicates an alteration of the wild-type gene. Alteration ofwild-type genes can also be detected by screening for alteration ofwild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 protein. For example,monoclonal antibodies immunoreactive with HERG can be used to screen atissue. Lack of cognate antigen would indicate a mutation. Antibodiesspecific for products of mutant alleles could also be used to detectmutant gene product. Such immunological assays can be done in anyconvenient formats known in the art. These include Western blots,immunohistochemical assays and ELISA assays. Any means for detecting analtered KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 protein can be used todetect alteration of wild-type KVLQT1, HERG, SCN5A, KCNE1 or KCNE2genes. Functional assays, such as protein binding determinations, can beused. In addition, assays can be used which detect KVLQT1, HERG, SCN5A,KCNE1 or KCNE2 biochemical function. Finding a mutant KVLQT1, HERG,SCN5A, KCNE1 or KCNE2 gene product indicates alteration of a wild-typeKVLQT1, HERG, SCN5A, KCNE1 or KCNE2 gene.

Mutant KVLQT1, HERG, SCN5A, KCNE1 or KCNE2 genes or gene products canalso be detected in other human body samples, such as serum, stool,urine and sputum. The same techniques discussed above for detection ofmutant genes or gene products in tissues can be applied to other bodysamples. By screening such body samples, a simple early diagnosis can beachieved for hereditary LQTS.

Initially, the screening method involves amplification of the relevantKVLQT1, HERG, SCN5A, KCNE1 or KCNE2 sequence. In another preferredembodiment of the invention, the screening method involves a non-PCRbased strategy. Such screening methods include two-step labelamplification methodologies that are well known in the art. Both PCR andnon-PCR based screening strategies can detect target sequences with ahigh level of sensitivity. Further details of these methods are brieflypresented below and further descriptions can be found in PCT publishedapplication WO 96/05306, incorporated herein by reference.

The most popular method used today is target amplification. Here, thetarget nucleic acid sequence is amplified with polymerases. Oneparticularly preferred method using polymerase-driven amplification isthe polymerase chain reaction (PCR). The polymerase chain reaction andother polymerase-driven amplification assays can achieve over amillion-fold increase in copy number through the use ofpolymerase-driven amplification cycles. Once amplified, the resultingnucleic acid can be sequenced or used as a substrate for DNA probes.

When the probes are used to detect the presence of the target sequences,the biological sample to be analyzed, such as blood or serum, may betreated, if desired, to extract the nucleic acids. The sample nucleicacid may be prepared in various ways to facilitate detection of thetarget sequence; e.g. denaturation, restriction digestion,electrophoresis or dot blotting. The targeted region of the analytenucleic acid usually must be at least partially single-stranded to formhybrids with the targeting sequence of the probe. If the sequence isnaturally single-stranded, denaturation will not be required. However,if the sequence is double-stranded, the sequence will probably need tobe denatured. Denaturation can be carried out by various techniquesknown in the art.

Analyte nucleic acid and probe are incubated under conditions whichpromote stable hybrid formation of the target sequence in the probe withthe putative targeted sequence in the analyte. The region of the probeswhich is used to bind to the analyte can be made completelycomplementary to the targeted region of the genes. Therefore, highstringency conditions are desirable in order to prevent false positives.However, conditions of high stringency are used only if the probes arecomplementary to regions of the chromosome which are unique in thegenome. The stringency of hybridization is determined by a number offactors during hybridization and during the washing procedure, includingtemperature, ionic strength, base composition, probe length, andconcentration of formamide. Under certain circumstances, the formationof higher order hybrids, such as triplexes, quadraplexes, etc., may bedesired to provide the means of detecting target sequences.

Detection, if any, of the resulting hybrid is usually accomplished bythe use of labeled probes. Alternatively, the probe may be unlabeled,but may be detectable by specific binding with a ligand which islabeled, either directly or indirectly. Suitable labels, and methods forlabeling probes and ligands are known in the art, and include, forexample, radioactive labels which may be incorporated by known methods(e.g., nick translation, random priming or kinasing), biotin,fluorescent groups, chemiluminescent groups (e.g., dioxetanes,particularly triggered dioxetanes), enzymes, antibodies and the like.Variations of this basic scheme are known in the art, and include thosevariations that facilitate separation of the hybrids to be detected fromextraneous materials and/or that amplify the signal from the labeledmoiety. A number of these variations are well known.

As noted above, non-PCR based screening assays are also contemplated inthis invention. This procedure hybridizes a nucleic acid probe (or ananalog such as a methyl phosphonate backbone replacing the normalphosphodiester), to the low level DNA target. This probe may have anenzyme covalently linked to the probe, such that the covalent linkagedoes not interfere with the specificity of the hybridization. Thisenzyme-probe-conjugate-target nucleic acid complex can then be isolatedaway from the free probe enzyme conjugate and a substrate is added forenzyme detection. Enzymatic activity is observed as a change in colordevelopment or luminescent output resulting in a 10³-10⁶ increase insensitivity. For example, the preparation ofoligodeoxynucleotide-alkaline phosphatase conjugates and their use ashybridization probes are well known.

Two-step label amplification methodologies are known in the art. Theseassays work on the principle that a small ligand (such as digoxigenin,biotin, or the like) is attached to a nucleic acid probe capable ofspecifically binding the target gene. Allele specific probes are alsocontemplated within the scope of this example.

In one example, the small ligand attached to the nucleic acid probe isspecifically recognized by an antibody-enzyme conjugate. In oneembodiment of this example, digoxigenin is attached to the nucleic acidprobe. Hybridization is detected by an antibody-alkaline phosphataseconjugate which turns over a chemiluminescent substrate. In a secondexample, the small ligand is recognized by a second ligand-enzymeconjugate that is capable of specifically complexing to the firstligand. A well known embodiment of this example is the biotin-avidintype of interactions. Methods for labeling nucleic acid probes and theiruse in biotin-avidin based assays are well known.

It is also contemplated within the scope of this invention that thenucleic acid probe assays of this invention will employ a cocktail ofnucleic acid probes capable of detecting the gene or genes. Thus, in oneexample to detect the presence of KVLQT1 in a cell sample, more than oneprobe complementary to KVLQT1 is employed and in particular the numberof different probes is alternatively 2, 3, or 5 different nucleic acidprobe sequences. In another example, to detect the presence of mutationsin the KVLQT1 gene sequence in a patient, more than one probecomplementary to KVLQT1 is employed where the cocktail includes probescapable of binding to the allele-specific mutations identified inpopulations of patients with alterations in KVLQT1. In this embodiment,any number of probes can be used.

Large amounts of the polynucleotides of the present invention may beproduced by replication in a suitable host cell. Natural or syntheticpolynucleotide fragments coding for a desired fragment will beincorporated into recombinant polynucleotide constructs, usually DNAconstructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the polynucleotide constructswill be suitable for replication in a unicellular host, such as yeast orbacteria, but may also be intended for introduction to (with and withoutintegration within the genome) cultured mammalian or plant or othereukaryotic cell lines. The purification of nucleic acids produced by themethods of the present invention are described, e.g., in Sambrook etal., 1989 or Ausubel et al., 1992.

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method described byBeaucage and Caruthers (1981) or the triester method according toMatteucci and Caruthers (1981) and may be performed on commercial,automated oligonucleotide synthesizers. A double-stranded fragment maybe obtained from the single-stranded product of chemical synthesiseither by synthesizing the complementary strand and annealing the strandtogether under appropriate conditions or by adding the complementarystrand using DNA polymerase with an appropriate primer sequence.

Polynucleotide constructs prepared for introduction into a prokaryoticor eukaryotic host may comprise a replication system recognized by thehost, including the intended polynucleotide fragment encoding thedesired polypeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome-binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Such vectors may be preparedby means of standard recombinant techniques well known in the art anddiscussed, for example, in Sambrook et al. (1989) or Ausubel et al.(1992).

An appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and may include, whenappropriate, those naturally associated with the KVLQT1 or other gene.Examples of workable combinations of cell lines and expression vectorsare described in Sambrook et al. (1989) or Ausubel et al. (1992); seealso, e.g., Metzger et al. (1988). Many useful vectors are known in theart and may be obtained from such vendors as Stratagene, New EnglandBiolabs, Promega Biotech, and others. Promoters such as the trp, lac andphage promoters, tRNA promoters and glycolytic enzyme promoters may beused in prokaryotic hosts. Useful yeast promoters include promoterregions for metallothionein, 3-phosphoglycerate kinase or otherglycolytic enzymes such as enolase or glyceraldehyde-3-phosphatedehydrogenase, enzymes responsible for maltose and galactoseutilization, and others. Vectors and promoters suitable for use in yeastexpression are further described in Hitzeman et al., EP 73,675A.Appropriate non-native mammalian promoters might include the early andlate promoters from SV40 (Fiers et al., 1978) or promoters derived frommurine Molony leukemia virus, mouse tumor virus, avian sarcoma viruses,adenovirus II, bovine papilloma virus or polyoma. Insect promoters maybe derived from baculovirus. In addition, the construct may be joined toan amplifiable gene (e.g., DHFR) so that multiple copies of the genemaybe made. For appropriate enhancer and other expression controlsequences, see also Enhancers and Eukaryotic Gene Expression, ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1983). See also, e.g.,U.S. Pat. Nos. 5,691,198; 5,735,500; 5,747,469 and 5,436,146.

While such expression vectors may replicate autonomously, they may alsoreplicate by being inserted into the genome of the host cell, by methodswell known in the art.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for survival or growth of a hostcell transformed with the vector. The presence of this gene ensuresgrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that a) confer resistance to antibioticsor other toxic substances, e.g. ampicillin, neomycin, methotrexate,etc., b) complement auxotrophic deficiencies, or c) supply criticalnutrients not available from complex media, e.g., the gene encodingD-alanine racemase for Bacilli. The choice of the proper selectablemarker will depend on the host cell, and appropriate markers fordifferent hosts are well known in the art.

The vectors containing the nucleic acids of interest can be transcribedin vitro, and the resulting RNA introduced into the host cell bywell-known methods, e.g., by injection (see, Kubo et al. (1988)), or thevectors can be introduced directly into host cells by methods well knownin the art, which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome); and other methods.See generally, Sambrook et al. (1989) and Ausubel et al. (1992). Theintroduction of the polynucleotides into the host cell by any methodknown in the art, including, inter alia, those described above, will bereferred to herein as “transformation.” The cells into which have beenintroduced nucleic acids described above are meant to also include theprogeny of such cells.

Large quantities of the nucleic acids and polypeptides of the presentinvention may be prepared by expressing the KVLQT1 nucleic acid orportions thereof in vectors or other expression vehicles in compatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, or amphibian or avian species, mayalso be useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. See,Jakoby and Pastan (eds.) (1979). Examples of commonly used mammalianhost cell lines are VERO and HeLa cells, Chinese hamster ovary (CHO)cells, and W138, BHK, and COS cell lines, although it will beappreciated by the skilled practitioner that other cell lines may beappropriate, e.g., to provide higher expression, desirable glycosylationpatterns, or other features. An example of a commonly used insect cellline is SF9.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker maybe on the same or a different DNA molecule,preferably the same DNA molecule. In prokaryotic hosts, the transformantmay be selected, e.g., by resistance to ampicillin, tetracycline orother antibiotics. Production of a particular product based ontemperature sensitivity may also serve as an appropriate marker.

Prokaryotic or eukaryotic cells transformed with the polynucleotides ofthe present invention will be useful not only for the production of thenucleic acids and polypeptides of the present invention, but also, forexample, in studying the characteristics of KVLQT1 or otherpolypeptides.

The probes and primers based on the KVLQT1 or other gene sequencesdisclosed herein are used to identify homologous KVLQT1 or other genesequences and proteins in other species. These gene sequences andproteins are used in the diagnostic/prognostic, therapeutic and drugscreening methods described herein for the species from which they havebeen isolated.

The studies described in the Examples below resulted in thedetermination of many novel mutations. Previous studies had defined 126distinct disease causing mutations in the LQTS genes KVLQT1, HERG,SCN5A, KCNE1 and KCNE2 (Wang Q. et al., 1996a; Curran et al., 1995; Wanget al., 1995a; Splawski et al., 1997a; Abbott et al., 1999; Chouabe etal., 1997; Wollnik et al., 1997; Neyroud et al., 1997; Splawski et al.,1997b; Tyson et al., 1997; Schulze-Bahr et al., 1997; Priori et al.,1999; Splawski et al., 1998; Wang et al., 1995b; Russell et al., 1996;Neyroud et al., 1998; Neyroud et al., 1999; Donger et al., 1997; Tanakaet al., 1997; Jongbloed et al., 1999; Priori et al., 1998; Itoh et al.,1998a; Itoh et al., 1998b; Mohammad-Panah et al., 1999; Saarinen et al.,1998; Ackerman et al., 1998; Berthet et al., 1999; Kanters, 1998; vanden Berg et al., 1997; Dausse et al., 1996; Benson et al., 1996; Akimotoet al., 1998; Satler et al., 1996; Satler et al., 1998; Makita et al.,1998, An et al., 1998; Schulze-Bahr et al., 1995; Duggal et al., 1998;Chen Q. et al., 1999; Li et al., 1998; Wei et al., 1999; Larsen et al.,1999a; Bianchi et al., 1999; Ackerman et al., 1999a; Ackerman et al.,1999b; Murray et al., 1999; Larsen et al., 1999b; Yoshida et al., 1999;Wattanasirichaigoon et al., 1999; Bezzina et al., 1999; Hoorntje et al.,1999). The sequence of each wild-type gene has been published. TheKVLQT1 can be found in Splawski et al. (1998) and the coding region ofthe cDNA is shown herein as SEQ ID NO:1 and the encoded KVLQT1 is shownas SEQ ID NO:2. SCN5A was reported by Gellens et al. (1992) and itssequence is provided by GenBank Accession No. NM_(—)000335. The codingsequence of SCN5A is shown herein as SEQ ID NO:3 and the encoded SCN5Ais shown as SEQ ID NO:4. Most of the mutations were found in KVLQT1(Yoshida et al., 1999) and HERG (Itoh et al., 1998b), and fewer in SCN5A(Wang Q. et al., 1996a), KCNE1 (Jiang et al., 1994) and KCNE2 (Ward,1964). These mutations were identified in regions with known intron/exonstructure, primarily the transmembrane and pore domains. In this study,we screened 262 individuals with LQTS for mutations in all knownarrhythmia genes. We identified 134 mutations, 80 of which were novel.Together with 43 mutations reported in our previous studies, we have nowidentified 177 mutations in these 262 LQTS individuals (68%). Thefailure to identify mutations in 32% of the individuals may result fromphenotypic errors, incomplete sensitivity of SSCP or presence ofmutations in regulatory sequences. However, it is also clear thatadditional LQTS genes await discovery (Jiang et al., 1994; Schott etal., 1995).

Missense mutations were most common (72%), followed by frameshiftmutations (10%), in-frame deletions, nonsense and splice site mutations(5-7% each). Most mutations resided in intracellular (52%) andtransmembrane (30%) domains; 12% were found in pore and 6% inextracellular segments. One hundred one of the 129 distinct LQTSmutations (78%) were identified in single families or individuals. Mostof the 177 mutations were found in KVLQT1 (75 or 42%) and HERG (80 or45%). These two genes accounted for 87% of the identified mutations,while mutations in SCN5A (14 or 8%), KCNE1 (5 or 3%) and KCNE2 (3 or 2%)accounted for the other 13%.

Multiple mutations were found in regions encoding S5, S5/P, P and S6 ofKVLQT1 and HERG. The P region of potassium channels forms the outer poreand contains the selectivity filter (Doyle et al., 1998). Transmembranesegment 6, corresponding to the inner helix of KcsA, forms the inner ⅔of the pore. This structure is supported by the S5 transmembranesegment, corresponding to the outer helix of KcsA, and is conserved fromprokaryotes to eukaryotes ((MacKinnon et al., 1998). Mutations in theseregions will likely disrupt potassium transport. Many mutations wereidentified in the C-termini of KVLQT1 and HERG. Changes in theC-terminus of HERG could lead to anomalies in tetramerization as it hasbeen proposed that the C-terminus of eag, which is related to HERG, isinvolved in this process (Ludwig et al, 1994).

Multiple mutations were also identified in regions that were differentfor KVLQT1 and HERG. In KVLQT1, multiple mutations were found in thesequences coding for the S2/S3 and S4/S5 linkers. Coexpression of S2/S3mutants with wild-type KVLQT1 in Xenopus oocytes led to simple loss offunction or dominant-negative effect without significantly changing thebiophysical properties of I_(Ks) channels (Chouabe et al., 1997; Shalabyet al., 1997; Wang et al., 1999). On the other hand, S4/S5 mutationsaltered the gating properties of the channels and modified KVLQT1interactions with minK subunits (Wang et al., 1999; Franqueza et al.,1999). In HERG, more than 20 mutations were identified in theN-terminus. HERG channels lacking this region deactivate faster andmutations in the region had a similar effect (Chen J. et al., 1999).

Mutations in KCNE1 and KCNE2, encoding minK and MiRP 1, the respectiveI_(Ks) and I_(Kr) β-subunits, altered the biophysical properties of thechannels (Splawski et al., 1997a; Abbott et al., 1999; Sesti andGoldstein, 1998). A MiRPI mutant, involved in clarithromyocin-inducedarrhythmia, increased channel blockade by the antibiotic (Abbott et al.,1999). Mutations in SCN5A, the sodium channel α-subunit responsible forcardiac I_(Na), destabilized the inactivation gate causing delayedchannel inactivation and dispersed reopenings (Bennett et al., 1995;Dumaine et al., 1996; Wei et al., 1999; Wang D W et al., 1996). OneSCN5A mutant affected the interactions with the sodium channel β-subunit(An et al., 1998).

It is interesting to note that probands with KCNE1 and KCNE2 mutationswere older and had shorter QTc than probands with the other genotypes.The significance of these differences is unknown, however, as the numberof probands with KCNE1 and KCNE2 genotypes was small.

This catalogue of mutations will facilitate genotype-phenotype analyses.It also has clinical implications for presymptomatic diagnosis and, insome cases, for therapy. Patients with mutations in KVLQT1, HERG, KCNE1and KCNE2, for example, may benefit from potassium therapy (Compton etal., 1996). Sodium channel blockers, on the other hand, might be helpfulin patients with SCN5A mutations (Schwartz et al. (1995). Theidentification of mutations is of importance for ion channel studies aswell. The expression of mutant channels in heterologous systems canreveal how structural changes influence the behavior of the channel orhow mutations affect processing (Zhou et al., 1998; Furutani et al.,1999). These studies improve our understanding of channel function andprovide insights into mechanisms of disease. Finally, mutationidentification will contribute to the development of genetic screeningfor arrhythmia susceptibility.

The present invention is described by reference to the followingExamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques specifically described in the Examples wereutilized.

EXAMPLE 1 Ascertainment and Phenotyping

Individuals were ascertained in clinics from North America and Europe.Individuals were evaluated for LQTS based on QTc (the QT intervalcorrected for heart rate) and for the presence of symptoms. In thisstudy, we focused on the probands. Individuals show prolongation of theQT interval (QTc≧460 ms) and/or documented torsade de pointes,ventricular fibrillation, cardiac arrest or aborted sudden death.Informed consent was obtained in accordance with local institutionalreview board guidelines. Phenotypic data were interpreted withoutknowledge of genotype. Sequence changes altering coding regions orpredicted to affect splicing that were not detected in at least 400control chromosomes were defined as mutations. No changes except knownpolymorphisms were detected ina ny of the genes in the controlpopulation. This does not exclude the possibility that some mutationsare rare variants not associated with disease.

EXAMPLE 2 Mutational Analyses

To determine the spectrum of LQTS mutations, we used SSCP (Single StandConformation Polymorphism) and DNA sequence analyses to screen 262unrelated individuals with LQTS. Seventeen primer pairs were used toscreen KVLQT1 (Splawski et al., 1998), twenty-one primer pairs were usedfor HERG (Splawski et al., 1998) and three primer pairs were used forKCNE1 (Splawski et al., 1997a) and KCNE2 (Abbott et al., 1999).Thirty-three primer pairs (Wang Q. et al., 1996b) were used in SSCPanalysis to screen all SCN5A exons in 50 individuals with suspectedabnormalities in I_(Na). Exons 23-28, in which mutations were previouslyidentified, were screened in all 262 individuals.

Gender, age, QTc and presence of symptoms are summarized in Table 1. Theaverage age at ascertainment was 29 with a corrected QT interval of 492ms. Seventy-five percent had a history of symptoms and femalespredominated with an˜2:1 ratio. Although the numbers were small,corrected QT intervals for individuals harboring KCNE1 and KCNE2mutations were shorter at 457 ms. TABLE 1 Age, QTc, Gender and Presenceof Symptoms Age*, y QTc, ms Genotype (mean ± SD) Gender (F/M) (mean ±SD) Symptoms^(†) KVLQT1 32 ± 19 52/23 493 ± 45 78% HERG 31 ± 19 51/29498 ± 48 71% SCN5A 32 ± 24 8/6 511 ± 42 55% KCNE1 43 ± 16 3/2 457 ± 2540% KCNE2 54 ± 20 3/0 457 ± 05 67% unknown 25 ± 16 56/29 484 ± 46 81%all 29 ± 19 173/89  492 ± 47 75%*-age at ascertainment^(†)-symptoms include syncope, cardiac arrest or sudden death

The SSCP analyses revealed many mutations. KVLQT1 mutations associatedwith LQTS were identified in 52 individuals (FIG. 1 and Table 2). Twentyof the mutations were novel. HERG mutations were identified in 68 LQTSindividuals (FIG. 2 and Table 3). Fifty-two of these mutations werenovel. SCN5A mutations were identified in eight cases (FIG. 3 and Table4). Five of the mutations were novel. Three novel KCNE1 mutations wereidentified (FIG. 4 and Table 5) and three mutations were identified inKCNE2 (FIG. 5 and Table 6) (Abbott et al., 1999). None of the KVLQT1,HERG, SCN5A, KCNE1 and KCNE2 mutations was observed in 400 controlchromosomes. TABLE 2 Summary of All KVLQT1 Mutations* Number NucleotideCoding of Change^(†) Effect Position Exon familles^(‡) Study del211-219del71-73 N-terminus 1 1 Ackerman et al., 1999a A332G† Y111C N-terminus 11 This de1451-452 A150fs/132 S2 2 1 JLN Chen Q. et al., 1999 T470G F157CS2 1 1 Larsen et al., 1999a G477 + 1A M159sp S2 2 1 JLN, This; Donger etal., 1997 1 UK G477 + 5A M159sp S2 1 1 Ackerman et al., 1999b G478A†E160K S2 3 1 This del500-502 F167W/del S2 3 1 Wang Q. et al., 1996a G168G502A G168R S2 3 7 This; Splawski et al., 1998; Donger et al., 1997C520T R174C S2/S3 3 1 Donger et al., 1997 G521A† R174H S2/S3 3 1 ThisG532A A178T S2/S3 3 1 Tanaka et al., 1997 G532C A178P S2/S3 3 1 Wang Q.et al., 1996a G535A† G179S S2/S3 3 1 This A551C Y184S S2/S3 3 2 This;Jongbloed et al., 1999 G565A G189R S2/S3 3 3 Wang Q. et al., 1996a;Jongbloed et al., 1999 insG567-568 G189fs/94 S2/S3 3 1 (RW + JLN)Splawski et al., 1997b G569A R190Q S2/S3 3 2 Splawski et al., 1998;Donger et al., 1997 del572-576 L191fs/90 S2/S3 3 1 JLN, Tyson et al.,1997; 1 RW Ackerman et al., 1999b 2 (JLN + RW) G580C† A194P S2/S3 3 1This C674T S225L S4 4 2 This; Priori et al., 1999 G724A D242N S4/S5 5 1Itoh et al., 1998b C727T† R243C S4/S5 5 2 This G728A R243H S4/S5 5 1 JLNSaarinen et al., 1998 T742C† W248R S4/S5 5 1 This T749A L250H S4/S5 5 1Itoh et al., 1998a G760A V254M S4/S5 5 4 This; Wang Q. et al., 1996a;Donger et al., 1997 G781A E261K S4/S5 6 1 Donger et al., 1997 T797C†L266P S5 6 1 This G805A G269S S5 6 1 Ackerman et al., 1999b G806A G269DS5 6 3 This; Donger et al., 1997 C817T L273F S5 6 2 This; Wang Q. etal., 1996a A842G Y281C S5 6 1 Priori et al., 1999 G898A A300T S5/Pore 61 Priori et al., 1998 G914C W305S Pore 6 1 JLN Chouabe et al., 1997G916A G306R Pore 6 1 Wang Q. et al, 1996a de1921- V307sp Pore 6 1 Li etal., 1998 (921 + 2) G921 + 1T† V307sp Pore 6 1 This A922-2C† V307sp Pore7 1 This G922-1C V307sp Pore 7 1 Murray et al., 1999 C926G T309R Pore 71 Donger et al., 1997 G928A† V310I Pore 7 1 This C932T T311I Pore 7 1Saarinen et al., 1998 C935T T312I Pore 7 2 This; Wang Q. et al., 1996aC939G I313M Pore 7 1 Tanaka et al., 1997 G940A G314S Pore 7 7 Splawskiet al., 1998; Russell et al., 1996; Donger et al., 1997; Jongbloed etal., 1999; Itoh et al., 1998b A944C Y315S Pore 7 3 Donger et al., 1997;Jongbloed et al., 1999 A944G Y315C Pore 7 2 Priori et al., 1999;Splawski et al., 1998 G949A D317N Pore 7 2 Wollnik et al., 1997;Saarinen et al., 1998 G954C K318N Pore 7 1 Splawski et al., 1998 C958GP320A Pore 7 1 Donger et al., 1997 G973A G325R S6 7 4 This; Donger etal., 1997; Tanaka et al., 1997 del1017-1019 delF340 S6 7 2 This;Ackerman et al., 1998 C1022A A341E S6 7 5 This; Wang Q. et al., 1996a;Berthet et al., 1999 C1022T A341V S6 7 7 This; Wang Q. et al., 1996a;Russell et al., 1996; Donger et al., 1997; Li et al., 1998 C1024T L342FS6 7 1 Donger et al., 1997 C1031T A344V S6 7 1 Donger et al., 1997G1032A A344sp S6 7 9 This; Kanters, 1998; Li et al., 1998; Ackerman etal., 1999b; Murray et al., 1999 G1032C A344sp S6 7 1 Murray et al, 1999G1033C G345R S6 8 1 van den Berg et al., 1997 G1034A G345E S6 8 1 WangQ. et al., 1996a C1046G† S349W S6 8 1 This T1058C L353P S6 8 1 Splawskiet al., 1998 C1066T† Q356X C-terminus 8 1 This C1096T R366W C-terminus 81 Splawski et al., 1998 G1097A† R366Q C-terminus 8 1 This G1097C R366PC-terminus 8 1 Tanaka et al., 1997 G1111A A371T C-terminus 8 1 Donger etal., 1997 T1117C S373P C-terminus 8 1 Jongbloed et al., 1999 C1172T†T391I C-terminus 9 1 This T1174C W392R C-terminus 9 1 Jongbloed et al.,1999 C1343G† P448R C-terminus 10 2 This C1522T R518X C-terminus 12 1JLN, This; Larsen et al., 1999 3 RW G1573A A525T C-terminus 12 1 Larsenet al., 1999b C1588T† Q530X C-terminus 12 1 JLN, This 1 RW C1615T R539WC-terminus 13 1 Chouabe et al., 1997 de16/ins7 E543fs/107 C-terminus 131 JLN Neyroud et al., 1997 C1663T R555C C-terminus 13 3 Donger et al.,1997 C1697T† S566F C-terminus 14 3 This C1747T† R583C C-terminus 15 1This C1760T T587M C-terminus 15 1 JLN, Donger et al., 1997; 1 RW Itoh etal., 1998b G1772A R591H C-terminus 15 1 Donger et al., 1997 G1781A†R594Q C-terminus 15 3 This del1892-1911 P630fs/13 C-terminus 16 1 JLNDonger et al., 1997 insC1893-1894 P631fs/19 C-terminus 16 1 Donger etal., 1997*—ins denotes insertion; del denotes deletion; sp denotes the lastunaffected amino acid before the predicted splice mutation; fs denotesthe last amino acid unaffected by a frameshift, following fs is thenumber of amino acids before termination; X denotes a stop codonoccurred.†—denotes novel mutation^(‡)—Number of Romano-Ward families unless otherwise indicated (UK—unknown)

TABLE 3 Summary of All HERG Mutations* Number Nucleotide Coding of RWChange Effect Position Exon Families Study C87A† F29L N-terminus 2 1This A98C† N33T N-terminus 2 2 This C132A† C44X N-terminus 2 1 ThisG140T† G47V N-terminus 2 1 This G157C† G53R N-terminus 2 1 This G167A†R56Q N-terminus 2 1 This T196G† C66G N-terminus 2 1 This A209G† H70RN-terminus 2 2 This C215A† P72Q N-terminus 2 2 This del221-251† R73fs/31N-terminus 2 1 This G232C† A78P N-terminus 2 1 This dupl234-250†A83fs/37 N-terminus 2 1 This C241T† Q81X N-terminus 2 1 This T257G† L86RN-terminus 2 1 This insC422-423† P141fs/2 N-terminus 3 1 ThisinsC453-454† P151fs/ N-terminus 3 1 This 179 dupl558-600 L200fs/N-terminus 4 1 Hoorntje et al., 1999 144 insC724-725† P241fs/89N-terminus 4 1 This del885† V295fs/63 N-terminus 4 1 This C934T† R312CN-terminus 5 1 This C1039T† P347S N-terminus 5 1 This G1128A† Q376spN-terminus 5 1 This A1129-2G† Q376sp N-terminus 6 1 This del1261Y420fs/12 S1 6 1 Curran et al., 1995 C1283A S428X S1/S2 6 1 Priori etal., 1999 C1307T T436M S1/S2 6 1 Priori et al., 1999 A1408G N470D S2 6 1Curran et al., 1995 C1421T T474I S2/S3 6 1 Tanaka et al., 1997 C1479GY493X S2/S3 6 1 Itoh et al., 1998a del1498-1524 del500-508 S3 6 1 Curranet al., 1995 G1592A† R531Q S4 7 1 This C1600T R534C S4 7 1 Itoh et al.,1998a T1655C† L552S S5 7 1 This delT1671 T556fs/7 S5 7 1 Schulze-Bahr etal., 1995 G1672C A558P S5 7 1 Jongbloed et al., 1999 G1681A A561T S5 7 4This; Dausse et al., 1996 C1682T A561V S5 7 4 This; Curran et al., 1995;Priori et al., 1999 G1714C G572R S5/Pore 7 1 Larsen et al., 1999a G1714TG572C S5/Pore 7 1 Splawski et al., 1998 C1744T R582C S5/Pore 7 1Jongbloed et al., 1999 G1750A† G584S S5/Pore 7 1 This G1755T† W585CS5/Pore 7 1 This A1762G N588D S5/Pore 7 1 Splawski et al., 1998 T1778C†I593T S5/Pore 7 1 This T1778G I593R S5/Pore 7 1 Benson et al., 1996G1801A G601S S5/Pore 7 1 Akimoto et al., 1998 G1810A G604S S5/Pore 7 2This; Jongbloed et al., 1999 G1825A† D609N S5/Pore 7 1 This T1831C Y611HS5/Pore 7 1 Tanaka et al., 1997 T1833 (A or Y611X S5/Pore 7 1Schulze-Bahr et al., 1995 G) G1834T V612L Pore 7 1 Satler et al., 1998C1838T T613M Pore 7 4 This; Jongbloed et aL, 1999 C1841T A614V Pore 7 6Priori et al., 1999; Splawski et al., 1998; Tanaka et al., 1997, Satleret al., 1998 C1843G† L615V Pore 7 1 This G1876A† G626S Pore 7 1 ThisC1881G† F627L Pore 7 1 This G1882A G628S Pore 7 2 This; Curran et al.,1995 A1885G N629D Pore 7 1 Satler et al., 1998 A1886G N629S Pore 7 1Satler et al., 1998 C1887A N629K Pore 7 1 Yoshida et al., 1999 G1888CV630L Pore 7 1 Tanaka et al., 1997 T1889C V630A Pore 7 1 Splawski etal., 1998 C1894T† P632S Pore 7 1 This A1898G N633S Pore 7 1 Satler etal., 1998 A1912G† K638E S6 7 1 This del1913-1915† delK638 S6 7 1 ThisC1920A F640L S6 7 1 Jongbloed et al., 1999 A1933T† M645L S6 7 1 Thisdel1951-1952 L650fs/2 S6 8 1 Itoh et al., 1998a G2044T† E682X S6/cNBD 81 This C2173T Q725X S6/cNBD 9 1 Itoh et al., 1998a insT2218-2219†H739fs/63 S6/cNBD 9 1 This C2254T† R752W S6/cNBD 9 1 This dupl2356-2386V796fs/22 cNBD 9 1 Itoh et al., 1998a del2395† I798fs/10 cNBD 9 1 ThisG2398 + 1C L799sp cNBD 9 2 This; Curran et al., 1995 T2414C† F805S cNBD10 1 This T2414G† F805C cNBD 10 1 This C2453T S818L cNBD 10 1 Berthet etal., 1999 G2464A V822M cNBD 10 2 Berthet et al., 1999; Satler et al.,1996 C2467T† R823W cNBD 10 2 This A2582T† N861I C-terminus 10 1 ThisG2592 + 1A D864sp C-terminus 10 2 This; Berthet et al., 1999 del2660†K886fs/85 C-terminus 11 1 This C2750T† P917L C-terminus 12 1 Thisdel2762† R920fs/51 C-terminus 12 1 This C2764T† R922W C-terminus 12 1This insG2775-2776† G925fs/13 C-terminus 12 1 This del2906† P968fs/4C-terminus 12 1 This del2959-2960† P986fs/ C-terminus 12 1 This 130C3040T† R1014X C-terminus 13 2 This de13094† G1031fs/ C-terminus 13 1This 24 insG3107-3108 G1036fs/ C-terminus 13 1 Berthet et al., 1999 82insC3303-3304† P1101fs C-terminus 14 1 This*—all characters same as in Table 2

TABLE 4 Summary of All SCN5A Mutations Number Nucleotide Coding of RWChange Effect Position Exon Families Study G3340A† D1114N DII/DIII 18 1This C3911T T1304M DIII/S4 22 1 Wattanasirichaigoon et al., 1999 A3974GN1325S DIII/S4/S5 23 1 Wang et al., 1995b C4501G† L1501V DIII/DIV 26 1This del4511-4519 del1505-1507 DIII/DIV 26 4 Wang et al., 1995a; Wang etal., 1995b del4850-4852† delF1617 DIV/S3/S4 28 1 This G4868A R1623QDIV/S4 28 2 This; Makita et al., 1998 G4868T† R1623L DIV/S4 28 1 ThisG4931A R1644H DIV/S4 28 2 This; Wang et al., 1995b C4934T T1645M DIV/S428 1 Wattanasirichaigoon et al., 1999 G5350A† E1784K C-terminus 28 2This; Wei et al., 1999 G5360A† S1787N C-terminus 28 1 This A5369G D1790GC-terminus 28 1 An et al., 1998 insTGA insD1795-1796 C-terminus 28 1Bezzina et al., 1999 5385-5386*—all characters same as in Table 2. Fifty individuals with suspectedabnormalities in I_(Na) were screened for all SCN5A exons. Allindividuals were screened for exons 23-28.

TABLE 5 Summary of All KCNE1 Mutations* Nucleotide Coding Number ofChange Effect Position Exon Families Study C20T T7I N-terminus 3 1 JLNSchulze- Bahr et al., 1997 G95A† R32H N-terminus 3 1 This G139T V47F S13 1 JLN Bianchi et al., 1999 TG151-152AT L51H S1 3 1 JLN Bianchi et al.,1999 A172C/TG TL58- S1 3 1 JLN Tyson et al., 176-177CT 59PP 1997 C221TS74L C-terminus 3 1 Splawski et al., 1997a G226A D76N C-terminus 3 1JLN, Splawski et 1 RW, al., 1997a; 1 (JLN + Tyson et al., RW) 1997;Duggal et al., 1998 T259C W87R C-terminus 3 1 Bianchi et al., 1999C292T† R98W C-terminus 3 1 This C379A† P127T C-terminus 3 1 This*—all characters same as in Table 2

TABLE 6 Summary of All KCNE2 Mutations Nucle- Number otide Coding ofChange Effect Position Exon Families Study C25G Q9E N-terminus 1 1Abbott et al., 1999 T161T M54T S1 1 1 Abbott et al., 1999 T170C I57T S11 1 Abbott et al., 1999

TABLE 7 Mutations by Type Type KVLQT1 HERG SCN5A KCNE1 KCNE2 TotalMissense 59 52 9 5 3 128 Nonsense 6 5 0 0 0 11 AA deletion* 2 2 5 0 0 9Frameshift 1 16 0 0 0 17 Splice 7 5 0 0 0 12 Total 75 80 14 5 3 177*—AA denotes amino acid

TABLE 8 Mutations by Position Gene Protein KVLQT1 HERG SCN5A KCNE1 KCNE2Position KVLQT1 HERG SCN5A minK MiRP1 Total Extracellular 0 7 1 1 1 10Trans- 33 13 5 0 2 53 membrane Pore 9 12 0 N/A N/A 21 Intracellular 3348 8 4 0 93 Total 75 80 14 5 3 177

While the invention has been disclosed in this patent application byreference to the details of preferred embodiments of the invention, itis to be understood that the disclosure is intended in an illustrativerather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

List of References

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1. An isolated DNA comprising a sequence of SEQ ID NO:1 as altered byone or more mutations selected from the group consisting of C1172T,C1343G, C 1588T, C1697T, C1747T and G1781A.
 2. An isolated nucleic acidprobe which hybridizes to the isolated DNA of claim 1 under conditionsat which it will not hybridize to wild-type KVLQT1 DNA.
 3. A method fordetecting a mutation in KVLQT1 said mutation selected from the groupconsisting of C1172T, C1343G, C1588T, C1697T, C1747T and G1781A whichcomprises analyzing a sequence of said KVLQT1 DNA or RNA from a humansample or analyzing the sequence of cDNA made from mRNA from said samplefor said mutation.
 4. The method of claim 3 wherein said mutation isdetected by a method selected from the group consisting of: a)hybridizing a probe specific for one of said mutations to RNA isolatedfrom said human sample and detecting the presence of a hybridizationproduct, wherein the presence of said product indicates the presence ofsaid mutation in the sample; b) hybridizing a probe specific for one ofsaid mutations to cDNA made from RNA isolated from said sample anddetecting the presence of a hybridization product, wherein the presenceof said product indicates the presence of said mutation in the sample;c) hybridizing a probe specific for one of said mutations to genomic DNAisolated from said sample and detecting the presence of a hybridizationproduct, wherein the presence of said product indicates the presence ofsaid mutation in the sample; d) amplifying all or part of said KVLQT1DNA in said sample using a set of primers to produce amplified nucleicacids and sequencing the amplified nucleic acids; e) amplifying part ofsaid KVLQT1 DNA in said sample using a primer specific for one of saidmutations and detecting the presence of an amplified product, whereinthe presence of said product indicates the presence of said mutation inthe sample; f) molecularly cloning all or part of said KVLQT1 DNA insaid sample to produce a cloned nucleic acid and sequencing the clonednucleic acid; g) amplifying said KVLQT1 DNA to produce amplified nucleicacids, hybridizing the amplified nucleic acids to a DNA probe specificfor one of said mutations and detecting the presence of a hybridizationproduct, wherein the presence of said product indicates the presence ofsaid mutation; h) forming single-stranded DNA from a KVLQT1 DNA fragmentof said gene from said human sample and single-stranded DNA from acorresponding fragment of a wild-type gene, electrophoresing saidsingle-stranded DNAs on a non-denaturing polyacrylamide gel andcomparing the mobility of said single-stranded DNAs on said gel todetermine if said single-stranded DNA from said sample is shiftedrelative to wild-type and sequencing said single-stranded DNA having ashift in mobility; i) forming a heteroduplex consisting of a firststrand of nucleic acid selected from the group consisting of a genomicDNA fragment isolated from said sample, an RNA fragment isolated fromsaid sample and a cDNA fragment made from mRNA from said sample and asecond strand of a nucleic acid consisting of a corresponding humanwild-type gene fragment, analyzing for the presence of a mismatch insaid heteroduplex, and sequencing said first strand of nucleic acidhaving a mismatch; j) forming single-stranded DNA from said KVLQT1 DNAof said human sample and from a corresponding fragment of an allelespecific for one of said mutations, electrophoresing saidsingle-stranded DNAs on a non-denaturing polyacrylamide gel andcomparing the mobility of said single-stranded DNAs on said gel todetermine if said single-stranded DNA from said sample is shiftedrelative to said allele, wherein no shift in electrophoretic mobility ofthe single-stranded DNA relative to the allele indicates the presence ofsaid mutation in said sample; and k) forming a heteroduplex consistingof a first strand of nucleic acid selected from the group consisting ofa genomic DNA fragment of said KVLQT1 DNA isolated from said sample, anRNA fragment isolated from said sample and a cDNA fragment made frommRNA from said sample and a second strand of a nucleic acid consistingof a corresponding gene allele fragment specific for one of saidmutations and analyzing for the presence of a mismatch in saidheteroduplex, wherein no mismatch indicates the presence of saidmutation.
 5. A method according to claim 4 wherein hybridization isperformed in situ.
 6. A method of assessing a risk in a human subjectfor long QT syndrome which comprises screening said subject for amutation in KVLQT1 selected from T3911 , P448R, Q530X, S566F, R583C andR594Q by comparing the sequence of said KVLQT1 or its expressionproducts isolated from a tissue sample of said subject with a wild-typesequence of said KVLQT1 or its expression products, wherein a mutationselected from T3911, P448R, Q530X, S566F, R583C and R594Q in thesequence of the subject indicates a risk for long QT syndrome.
 7. Themethod of claim 6 wherein said expression product is selected from mRNAof KVLQT1 DNA or a polypeptide encoded by said gene.
 8. The method ofclaim 6 wherein one or more of the following procedures is carried out:(a) observing shifts in electrophoretic mobility of single-stranded DNAfrom said sample on non-denaturing polyacrylamide gels; b) hybridizing aprobe to genomic DNA isolated from said sample under conditions suitablefor hybridization of said probe to said gene; (c) determininghybridization of an allele-specific probe to genomic DNA from saidsample; (d) amplifying all or part of said KVLQT1 DNA from said sampleto produce an amplified sequence and sequencing the amplified sequence;(e) determining by nucleic acid amplification the presence of a specificmutant allele in said sample; (f) molecularly cloning all or part ofsaid KVLQT1 DNA from said sample to produce a cloned sequence andsequencing the cloned sequence; (g) determining whether there is amismatch between molecules (1) said KVLQT1 DNA or mRNA isolated fromsaid sample, and (2) a nucleic acid probe complementary to the humanwild-type gene DNA, when molecules (1) and (2) are hybridized to eachother to form a duplex; (h) amplification of said KVLQT1 DNA sequence insaid sample and hybridization of the amplified sequence to nucleic acidprobes which comprise wild-type gene sequences; (i) amplification ofsaid KVLQT1 DNA sequence in said tissue and hybridization of theamplified sequence to nucleic acid probes which comprise mutant genesequence; (j) screening for a deletion mutation; (k) screening for apoint mutation; (l) screening for an insertion mutation; (m) determiningin situ hybridization of said KVLQT1 DNA in said sample with one or morenucleic acid probes which comprise said KVLQT1 DNA sequence or a mutantsequence of said KVLQT1 DNA; (n) immunoblotting; (o)immunocytochemistry; (p) assaying for binding interactions between saidprotein isolated from said tissue and a binding partner capable ofspecifically binding the polypeptide expression product of a mutantallele and/or a binding partner for the polypeptide and assaying for theinhibition of biochemical activity of said binding partner.
 9. A methodfor diagnosing a mutation which causes long QT syndrome comprisinghybridizing a probe which hybridizes to isolated DNA comprising asequence of SEQ ID NO:1 as altered by one or more mutations selectedfrom the group consisting of C1172T, C1343G, C1588T, C1697T, C1747T andG1781A and not to wild-type KVLQT1 DNA, to a patient's sample of DNA orRNA, the presence of a hybridization signal being indicative of long QTsyndrome.
 10. A method according to claim 9 wherein the patient's DNA orRNA has been amplified and said amplified DNA or RNA is hybridized withsaid probe.
 11. A method according to claim 9 wherein said hybridizationis performed in situ.
 12. A method according to claim 9 wherein saidassay is performed using nucleic acid microchip technology.
 13. A methodfor diagnosing a mutation which causes long QT syndrome comprisingamplifying a region of gene or RNA for KVLQT1 and sequencing theamplified gene or RNA wherein long QT syndrome is indicated by any oneor more mutations selected from the group consisting of C1172T, C1343G,C1588T, C1697T, C1747T and G1781A.
 14. A method for diagnosing amutation which causes long QT syndrome comprising identifying a mismatchbetween a patient's DNA or RNA and a wild-type DNA or RNA probe whereinsaid probe hybridizes to a region of DNA or RNA wherein said regioncomprises a mutation of SEQ ID NO:1 selected from the group consistingof C1172T, C1343G, C1588T, C1697T, C1747T and G1781A.
 15. The method ofclaim 14 wherein the mismatch is identified by an RNase assay.
 16. Anisolated DNA encoding a KVLQT1 polypeptide of SEQ ID NO:2 having amutation selected from the group consisting of T3911, P448R, Q530X,S566F, R583C and R594Q.